Method and apparatus for intracellular manipulation of a biological cell

ABSTRACT

A process is provided for the intracellular manipulation of a biological cell ( 3 ) which is positioned adhering to a support area ( 5 ) in a culture medium ( 2 ). Inside the support area ( 5 ) for the cell ( 3 ) an opening into the membrane of the cell ( 3 ) is created spaced from its support edge. The edge of the cell membrane surrounding the opening, adhering to the support area ( 5 ), thus seals off the cell fluid situated in the interior of the cell ( 3 ) from the culture medium ( 2 ) and insulates the cell fluid against the culture medium ( 2 ). The interior of the cell ( 3 ) is manipulated through the opening. An apparatus for implementing the process is also provided, including an object carrier ( 4 ) with a support area ( 5 ) for adhering the cell and a poration tool ( 6 ) for creating the opening in the cell membrane. The poration tool ( 6 ) may be any of various chemical, mechanical and/or electrical devices.

CROSS REFERENCE TO RELATED APPLICATION

[0001] This is a division of U.S. patent application Ser. No.09/321,051, filed May 27, 1999, now allowed.

BACKGROUND OF THE INVENTION

[0002] The invention concerns a method for intracellular manipulation ofat least one deposited biological cell situated in a cultural mediumadhering to a support area, wherein an opening is created in themembrane of the cell, and the cell interior is manipulated through thisopening. The invention furthermore relates to an apparatus formanipulation of the cell interior of at least one biological cell havinga cell membrane situated in a culture medium. The apparatus has anobject carrier, which has at least one support area on which the cell isadherently depositable, a poration tool for opening the cell membrane,and at least one entry channel situated in the area of the poration toolfor manipulation of the cell interior.

[0003] An apparatus of the type mentioned at the beginning is alreadyknown from Alberts, B. et al., Molecular Biology of the Cell, ThirdPrinting, VCH Verlag (1995), page 212 ff, which has a hollow needleconnected with a suction device and made of an electrical insulatingmaterial having an interior cavity which has an opening on the free endof the hollow needle. For opening the cell membrane, the openingsituated at the free end of the hollow needle is set upon the exteriorof the cell membrane in order then to create an underpressure in theinterior cavity of the hollow needle by means of the suction device.Through this underpressure, a cell membrane piece situated in front ofthe opening of the hollow needle is torn out of the membrane formation.After introducing the opening into the cell membrane the hollow needle,engaging the edge of the cell membrane surrounding the opening,electrically insulates the cell fluid contained in the interior of thecell against the culture medium. The interior of the cell is manipulatedthrough the opening introduced into the cell membrane. For example, thecell nucleus can be removed by sucking cell fluid into the hollow needlefrom the cell, and subsequently another cell nucleus can be installedthrough the opening into the cell interior.

[0004] The previously known process and the apparatus for implementingthe process have the disadvantage that a micromanipulator is necessaryfor positioning the hollow needle on the cell. This results in acomparatively more complicated and expensive device. Moreover, theaccessibility of the cells situated on the object carrier by themicromanipulator is greatly reduced. The process and the apparatus are,for this reason, only suited for an intracellular manipulation ofindividual, or at best for a simultaneous manipulation of a small numberof cells situated on the object carrrier. At the same time, a costly,manual positioning of the hollow needle on the cell is necessary.

[0005] Arranging a cell floating in a culture medium between two largesurface area electrodes, respectively spaced at a distance from thecell, and applying an electric voltage to these electrodes is alsoalready known from Alberts, B. et al., Molecular Biology of the Cell,Third printing, VCH Verlag (1995), page 213. Moreover, the membrane ofthe cell is simultaneously opened at several places by electroporationso that the genetic material situated in the culture medium to beintroduced into the cell can be diffused into the cell interior throughthe openings of the cell. In this connection, however, it isdisadvantageous that such an introduction of genetic material is subjectto statistical fluctuations and is influenced by various parameters suchas the concentration of genetic material in the area of the cell, thesize of the genetic material to be introduced, and the size of theopenings created in the cell membrane. The previously known processconsequently does allow any selective manipulation of the cell interior.

SUMMARY OF THE INVENTION

[0006] There thus exists an object of creating a process and anapparatus of the type mentioned at the beginning, which make a simplemanipulation of the interior of a cell possible. In particular, anexpensive manual positioning of a hollow needle on the cell to beprocessed is to be avoided. This objective is accomplished according tothe process of the invention in that the opening is introduced into thecell membrane within the support area of the cell and at a distance fromits support edge.

[0007] In this manner, it is possible to arrange a poration agent forcreating the cell membrane opening or a poration tool in the supportarea of the cell on the object carrier, so that when the cell isdeposited on the support area, it is at the same time positioned on theporation agent or the poration tool. In this manner, an expensive manualpositioning of a poration tool can be omitted. Since the opening isintroduced into the cell membrane within the support area of the celland at a distance from the edge of the support area, the membrane areaof the cell surrounding the opening and adhering to the support area forthe cell seals the opening against the culture medium. The cell fluidsituated in the interior of the cell is thereby electrically insulatedagainst the culture medium as extensively as possible, so that apotential equilization between the cell potential in the interior of thecell and that of the culture medium is prevented. Through the openingcreated into the cell membrane, the cell interior can be manipulated.

[0008] With an especially advantageous embodiment of the invention, theopening is introduced into the cell membrane by means ofelectroporation. While conducting the process, the electroporationelectrode can, for example, be arranged in the support area on which theadhering cell lies. To introduce the opening into the cell membrane,only an electrical voltage then needs to be applied between theelectroporation electrode and the culture medium. This brings about aflow of electrical current which opens the cell membrane.

[0009] With another embodiment of the invention, at least one mechanicalimpulse is exerted to introduce the opening into the cell membrane on apartial area of the cell membrane. In this connection, this partial areais released from the membrane formation. Optionally, an impulsesuccession can also be applied with several individual impulses.

[0010] It is especially advantageous if the opening is introduced intothe cell membrane by means of sound waves, especially ultrasound and/orhypersound waves. Here, it is even possible for the sound waves to befocused on the area of the cell membrane to be opened and/or for severalsound waves to be superimposed, such that their oscillations in the areaof the cell membrane are overlaid into an oscillation with increasedamplitude. The cell membrane can thereby be opened without contact.

[0011] A contact-free opening of the cell membrane can, however, alsotake place in that a partial area of the cell membrane is irradiatedwith energy-rich radiation, especially with laser radiation. In thisconnection, the wave length of the radiation is preferably selected suchthat the cell membrane absorbs the radiation well. Expediently, theradiation is launched into the cell at its support area. Optionally,however, a laser beam can also be launched into the cell outside thesupport area, in that first of all a small launch opening is introducedin a membrane area situated there. The laser beam is subsequentlyprojected through the opening and through the interior of the cell to amembrane area situated in the support area of the cell, in order to cutout a partial area of the membrane from the membrane formation byswivelling the laser beam around the launch opening.

[0012] With another embodiment of the process, the opening is introducedinto the cell membrane by the action of a chemical poration substance.Perforin or Triton®, for example, can be used as poration agents.

[0013] It is especially advantageous if an electrical and/or chemicaland/or a radiation-activatable chemical substance is used, and if thissubstance for introducing the opening into the cell membrane isactivated by the action of radiation, a chemical and/or an electricalfield. The substance is thus activated by supplying energy. In thismanner, free radicals can be generated, for example, which destroy thepartial area of the cell membrane to be opened. In the inactive state,the substance behaves largely neutrally toward the cell so that itpractically does not influence placing the cell on the support area. Achemical substance which is activated by administering a furthersubstance can also be used.

[0014] Another embodiment of the process provides that a partial area ofthe cell membrane to be opened is released from the membrane formationwith an underpressure and/or an overpressure. For this purpose, forexample, a small opening can be provided inside the support area on anobject carrier having the support area. Suction is applied through theopening to the cell so strongly that the membrane area situated in frontof the opening is torn out of the membrane formation. For opening thecell membrane, an overpressure impulse can also be exerted through theopening on a membrane area of the cell. It is advantageous for the cellto be fixed on the support area by the force of suction. In this mannerthe adhesion of the cell to the support area of the object carrier canbe improved. The force of suction is here so proportioned that the cellmembrane is not mechanically damaged by the force of the suction.

[0015] It is advantageous if after introducing the opening into the cellmembrane at least one substance and/or one cell component is removedfrom the cell interior and/or placed in the cell interior though theopening. Thus, for example, a medication, a protein and/or anotherbiologically active substance can be brought through the opening intothe interior of the cell in order to test its reaction. However, a geneor gene fragment can also be removed from the cell or introduced intoit. Even the cell nucleus can be removed from the cell through theopening and replaced by another.

[0016] With regard to the apparatus of the invention, the accomplishmentof the object mentioned above consists in that the poration tool isarranged within the support area. The cell can thereby be deposited onthe poration tool situated in the support area. Advantageously, anexpensive manual positioning of the poration tool on the cell is therebyavoided. Furthermore, no auxiliary devices, for examplemicromanipulators, are needed. It is thus possible to manipulateintracellularly several cells arranged tightly adjacent to one anotherin the support area at the same time. After introducing the opening intothe cell membrane, the edge of the cell membrane surrounding the openingremains in contact with the support area of the object carrier and sealsthe opening of the cell membrane against the culture medium

[0017] An especially advantageous embodiment of the invention providesthat the poration tool is bounded by at least one electrical insulatorwithin the support area. By means of the electrical insulator, the cellfluid situated in the interior of the cell is then well insulatedagainst the culture medium so that the interior of the living cell canbe manipulated over a longer period of time without the cell dying off.The insulation resistance is dependent upon the cell type and ispreferably greater than 10 megaohm. A potential equalization between theculture medium and the cell fluid is thereby prevented to the greatestextent. Optionally, the object carrier can consist wholly of aninsulating material. The insulator can also, however, be arranged in theinterior of the object carrier at a distance from the surface of thesupport area.

[0018] It is advantageous if the poration tool substantiallyconcentrically surrounds the opening of the passage channel situated inthe support area of the object carrier. The cell interior can then beeven better manipulated through the opening without the edge area of thecell membrane surrounding the opening being damaged by the manipulation.

[0019] It is especially advantageous if the poration tool is anelectroporation electrode, to which a reference electrode capable ofbeing brought into contact with the culture medium is allocated, and ifthe electroporation electrode and the reference electrode areconnectable for electroporation of the cell membrane with a source ofelectrical voltage. The electroporation electrode is arranged within thesupport area of the object carrier so that a cell adherently depositedon the insulator can likewise be deposited on the electrode, or at leastcan approach this up to the radius of action of an electrical fieldemanating from the electrode. When electroporation voltage is applied tothe electrode, an electric current flows which introduces an openinginto the cell membrane. At the same time, the edge of the cell membranesurrounding the opening remains in contact with the support area andseals off the opening against the culture medium. After creating theopening into the cell membrane, the electrode is separated from theelectroporation voltage source, so that the interior of the cell canthen be altered through the opening.

[0020] With one advantageous embodiment of the apparatus, the porationtool for opening the cell membrane is movable by means of an actuator,especially a piezo element, transversely to the surface of the supportarea relative to the object carrier. With this device, the opening isthus mechanically introduced into the cell membrane. The poration toolcan thus be alternatively moved toward and away from the opening on thecell membrane. For this purpose, the actuator is connected with anactivation facility for generating a mechanical vibration, especially anultrasound or hypersound vibration. The poration tool can be mobile in adirection running at right angles or obliquely to the surface of thesupport area relative to the object carrier.

[0021] It is advantageous if the poration tool has at least one sharptip or edge, preferably protruding relative to the surface plane of thesupport area. The poration tool can then optionally lie on the cellmembrane, so that this can be mechanically opened even better. If theporation tool is an electrode for electroporation of the cell membrane,there results an especially high electric field strength when a porationvoltage is applied to the electrode, which facilitates opening the cellmembrane.

[0022] It is especially advantageous if a laser beam is provided asporation tool, and if the radiation path of the laser beam is passedthrough the entry channel to the opening situated in the support area.With this device, a partial area of the cell membrane can be irradiatedfor a short time with energy-rich optical radiation, wherein the latteris so strongly heated that the cell membrane opens. Optionally, beamguidance means can be arranged inside or outside the entry channel.

[0023] It is especially advantageous if a laser diode is incorporatedinto the object carrier for generating the laser beam. In this manner,the laser diode can even be arranged directly behind the opening of theentry channel so that the laser radiation can be launched directly andconsequently largely free of loss into the cell membrane of the celldeposited on the support area.

[0024] It is provided with an advantageous embodiment of the inventionthat the poration tool for opening the cell membrane has a chemicalporation substance in the support area of the object carrier and/or thata chemical poration substance can be fed in through the entry channel toits opening. The opening can thus also be introduced chemically into thecell membrane, whereby Perforin or Triton® can be used as porationagents. Optionally, the entry channel can be connected or connectablewith a deposit containing the poration substance, from which theporation substance is feedable to the cell membrane.

[0025] With another embodiment, the device has at least one pump which,for opening the cell membrane by contacting with underpressure oroverpressure, is connected with the entry channel and/or the entrychannel is connectable with an underpressure or overpressure reservoirthrough a valve or similar shut-off element. The opening can beintroduced into the cell membrane through underpressure or overpressure,wherein the underpressure or overpressure is shut off following openingthe cell membrane. In this manner, sucking off cell fluid from theinterior of the cell during opening the cell membrane throughunderpressure is avoided to the greatest extent. Correspondingly, whenopening the cell membrane using overpressure, any medium situated in theentry channel (which is preferably a fluid) can be prevented fromreaching into the interior cell. For turning off the underpressure oroverpressure, a pressure change occurring in the entry channel whenopening the cell membrane can be determined. Advantageously, the entrychannel can be also used before depositing the cell, in order to suckoff culture medium from the support area, so that a current arises inthe culture medium which leads the cells situated therein to the openingof the entry channel arranged in the area of the poration tool.

[0026] An advantageous embodiment provides that the poration tool isarranged on a projection protruding in relation to the surface plane ofthe support area. In this manner, there results a good electrical and/ormechanical contact between the poration tool and the cell membrane.

[0027] Expediently, it is provided that the cross section of theprojection tapers at the furthest projecting point. The cell thenadheres especially well on the support area of the object carrier in thearea of the projection. Moreover, in terms of production engineering theinsulator can be better applied during production of the object carrieras a coating to the tapering area of the projection.

[0028] With an advantageous refinement of the invention, it is providedthat the object carrier has a contour in the support area, which has atleast one profile depression running around the poration tool and/or oneprofile projection running around the poration tool. In this manner, abetter sealing of the cell fluid against the culture medium by the cellmembrane adhering to the insulator is obtained.

[0029] It is advantageous if the profile depression and/or the profileprojection is interrupted by at least one gap in the projectiondirection. The cell can then adhere better in the area of the contouringto the surface of the object carrier. The profile projection or theprofile depression can, for example, have a honeycomb structure, or astructure in the manner of a checker or chess board pattern.

[0030] It is especially advantageous if the profile depression and/orprofile projection is constructed as a ring-shape, and if preferablyseveral such annular profile depressions and/or profile projections arearranged substantially concentrically to the poration tool.Consequently, several profile depressions and projections are connectedor inserted one after the other radially to the poration tool, so thatthe cell fluid is even better sealed off against the culture medium.

[0031] A preferred embodiment of the invention provides that theelectrical insulator is an insulation layer arranged on the surface ofthe contour. Advantageously, with the insulation layer contoured in thismanner, the path for a creeping current flowing on the surface of theinsulator from the cell fluid to the culture medium is enlarged, so thatthe cell fluid situated in the interior of the cell is even betterinsulated against the culture medium after opening the cell membrane.

[0032] Another embodiment provides that the profile projection(s) is(are) installed on the surface of the electrical insulator. The objectcarrier is then easier to manufacture in terms of assembly.

[0033] It is especially advantageous if a cell coating having at leastone cell adhesion protein and/or a hydrophilic coating and/orimmediately adjacent to the poration tool a hydrophobic coating isarranged in the support area of the object carrier. The cell membranethen adheres better to the object carrier. The cell adhesion coating caninclude, for example, laminin, fibronectin or poly-L-lysine. Optionally,a hydrophobic coating with binding sites for hydrophobic lipids found inthe cell membrane can also be arranged on the edge of the support areabordering on the electrode.

[0034] It is advantageous if, as a mechanical guide for the cells,boundary walls are arranged on both sides of the poration tool, whichpreferably delimit a groove-like guide channel. Moreover, the porationtool is preferably arranged in the middle between the boundary walls atthe bottom of the groove of the guide channel, so that cells situated inthe guide channel can essentially move only in the direction ofextension of the guide channel and then necessarily come into contactwith the poration tool.

[0035] It is advantageous if, for creating an electrical field leadingthe cell to the poration tool, at least one supplemental electrode isarranged in the support area and/or adjacent thereto. In this manner, anelectric field can be created on the surface of the object carrier,which exerts a force on the biological cells, their dielectricityconstants differing from those of the culture medium in which they arearranged, which leads the cells to the poration tool.

[0036] With a preferred embodiment of the invention, the object carrieris constructed approximately as a plate-shaped carrier element which hasthe support area on a flat side. The entry channel penetrates the objectcarrier proceeding from the support area to the reverse side of theobject carrier facing away therefrom, and the object carrier has a wallweakening or thinning (attenuation) in the area of the entry channel,which in the direction of extension of the entry channel has a smallerdimension than a wall area adjacent to the wall attenuation. The entrychannel is thus passed through the object carrier in the area of thewall attenuation and penetrates this, preferably at right angles to theplane stretching across the support area. In this manner, an especiallyshort entry channel is obtained. The entry channel can, for example, beconnected with a pump or a suction facility arranged on the reverse sideof the object carrier. The wall area of the object carrier bordering onthe wall attenuation, and preferably encircling it, has a greater wallthickness than the wall attenuation which improves the mechanicalstability of the object carrier.

[0037] It is advantageous if the wall attenuation is arranged on apreferably funnel-shaped form formation in situated on the reverse sideof the object carrier facing away from the support area. The supportarea can then be arranged in a plane extending up to and over the wallattenuation, so that the cells can better accumulate thereon.

[0038] An especially advantageous refinement of the invention providesthat, in the support area, several poration tools having at least oneentry channel are preferably arranged as arrays. With such a device, aplurality of cells closely adjacent to one another can beintracellularly manipulated, either at the same time or successively,whereby statistical fluctuations can be eliminated. Optionally, amultiplexer can be incorporated into the object carrier with which aplurality of electroporation electrodes can be controlled alterativelyone after the other, whereby the number of supply leads to the objectcarrier is correspondingly reduced.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

[0039] The foregoing summary, as well as the following detaileddescription of preferred embodiments of the invention, will be betterunderstood when read in conjunction with the appended drawings. For thepurpose of illustrating the invention, there is shown in the drawingsembodiments which are presently preferred. It should be understood,however, that the invention is not limited to the precise arrangementsand instrumentalities shown.

[0040] In the drawings:

[0041]FIG. 1 is a longitudinal section through an apparatus of theinvention for manipulation of the interior of a cell, having an objectcarrier with a poration tool, on which carrier a biological cellsituated in a culture medium is adherently deposited;

[0042]FIG. 2 is a longitudinal section through an apparatus similar toFIG. 1 wherein, however, the support area of the object carrier isuncontoured;

[0043]FIG. 3 is a plan view of the poration tool shown in FIG. 2;

[0044]FIG. 4 is a representation similar to FIG. 2 wherein, however, theporation tool is constructed as an electrode having a substantiallycylindrical shape;

[0045]FIG. 5 is a plan view of the electrode in accordance with FIG. 4;

[0046]FIG. 6 is a longitudinal section through an apparatus in which theporation tool is movable by means of a piezo element;

[0047]FIG. 7 is a longitudinal section through an apparatus in which alaser beam is launched into the entry channel;

[0048]FIG. 8 is a longitudinal section through an object carrier whichhas a poration tool arranged between two boundary walls inside a surfacestructuring;

[0049]FIG. 9 is a plan view on an object carrier which has an array witha plurality of poration tools and entry channels;

[0050]FIG. 10 is a plan view on an object carrier whose support area hasa checkered structure; and

[0051]FIG. 11 is a plan view on an object carrier whose support area hasa honeycomb structure.

DETAILED DESCRIPTION OF THE INVENTION

[0052] An apparatus, designated overall with 1, for manipulation of theinterior of a biological cell 3 situated in a culture medium 2 (FIG. 1)has an object carrier 4 which has a support area 5 on which the cell 3can be adherently deposited. The cell 3 is thus immobilized on theobject carrier 4 and adheres to the support area 5. For creating anopening into the cell 3 adhering to the support area 5, a poration tool6 is arranged inside the support area 5. The object carrier 4 has aentry channel 7 for manipulation of the cell interior which opens intothe support area 5 in the area of the poration tool 6. In the operatingposition, the edge of the support area 4 encircling the opening isarranged at a distance from the support edge of the cell 3. The membraneof the cell 3 adhering to the object carrier 4 thus encircles theopening of the entry channel 7, through which the cell fluid situated inthe cell interior is sealed off against the culture medium 2 aftercreating the opening into the cell membrane.

[0053] With the embodiments according to FIGS. 1 to 3, the poration tool6 is an electroporation electrode which has an active electrode area 8projecting in relation to the surface plane of the support area 5. Theelectrode is constructed as a hollow electrode which is penetrated bythe entry channel 7. A segment of the entry channel 7 extends from theend of the electrode facing away from the active electrode area 8 to theactive electrode end 8. As is especially well recognizable in FIGS. 3and 5, the active electrode area 8 encircles annularly the opening ofthe entry channel 7. Furthermore, an electrical insulator 9 surroundsthe active electrode area 8 in the support area 5 on which the cell 3 isdepositable sealed off against the culture medium 2.

[0054] The electrode is connected by means of a conductor pathincorporated into the object carrier to an electric or electroniccircuit element with which it is connectable with an electroporationvoltage element. For opening the cell membrane, an electrical voltage isapplied between the poration tool 6 and the culture medium 2, whereuponan electrical current flows into the cell membrane through the porationtool 6, which opens the cell membrane in the area of the poration tool6. The opening introduced into the cell 3 by means of the poration tool6 is sealed off against the culture medium 2 by the cell membrane areaadhering to the insulator 9 of the object carrier 4. In this manner, apotential equalization between the potential of the cell fluid situatedin the interior of the cell 3 and that of the culture medium 2 isprevented.

[0055] After opening the cell membrane, the interior of the cell 3 ismanipulated through the entry channel 7. Here, for example, a substancecan be removed from the cell interior and/or introduced into the cellinterior through the entry channel 7. The substance can be, for example,a cell component, a gene, a protein, a toxic substance which the cellshould detect, and/or a medication whose action on the cell should beexamined, or with which the cell should be treated. Substances presentin the cell or cell components can also be altered, in that they are,for example, irradiated through the entry channel 7 with energy-richradiation. A micromanipulator or similar tool can also be introducedinto the cell interior through the entry channel 7 to manipulate thecell interior there mechanically. In this manner, a change can beundertaken selectively at a certain place in the cell interior.

[0056] In the embodiments according to FIGS. 1 and 3, the poration tool6 constructed as an electrode assumes approximately the shape of a conicsection, wherein the substantially cylindrical entry channel 7penetrates along the central axis of the cone. The symmetry axis of theelectrode is at all times approximately at right angles to the surfaceplane of the object carrier 4 in the support area 5. The poration tool 6can be wholly or partially sunk into the surface of the object carrier4.

[0057] The active electrode region 8 of the electrode respectivelysurrounds the opening of the entry channel 7 and has a sharp ring edgeprojecting in relation to the surface plane of the support area 5, thecross section of which tapers proceeding from the surface plane of theobject carrier to the furthest projecting point of the electrode. Inthis manner, an especially high electrical field intensity arises whenan electroporation voltage is applied to the poration tool 6 in theactive electrode region 8, which facilitates opening the cell membrane.

[0058] The portion of the entry channel 7 which penetrates the hollowelectrode can be filled with culture medium 2. In this manner, theelectric charge carrier can reach from the inner wall of the electrodesurrounding the entry channel 7 to the cell membrane situated on theopening of the entry channel 7, whereby a greater active electrodesurface results overall, and the electrical contact resistance betweenthe electrode and the cell fluid is correspondingly diminished. Theelectrode surface can have a surface roughness which enlarges thesurface of the electrode. The electrode can, for example, be made ofporous silicon or have a coating of this or another porous material.

[0059] As is especially well recognizable in FIGS. 1, 4, 6 and 7, theelectrical insulator 9 has a projection 11 inside the support area 5protruding in relation to its surface plane, on whose free end facingaway from the surface plane the active electrode area 8 of theelectroporation electrode is arranged. The active electrode area 8 isthereby electrically well-connected to the cell 3 adhering to thesupport area. The cross section of the projection 11 tapers proceedingfrom the surface plane of the support area 5 to the furthest protrudingpoint. In this manner, the projection 11 and the electrode can be betterproduced in terms of manufacturing technology. Moreover, the taperingprojection 11 makes possible a good mechanical stability. The projection11 can, however, also have a constant cross section in the direction ofits extension. The projection 11 can, for example, also be manufacturedusing LIGA technology.

[0060] The object carrier 4 has a subatantially plate-shaped substrate12 which, for example, can be made of a semiconductor material (forexample silicon or gallium arsenide), silicon carbide, glass or plastic.On this substrate, the insulator 9 can be applied as a coating, forexample by sputtering. Optionally, the substrate 12 can also be aflexible sheet.

[0061] In the embodiment according to FIG. 6, a piezo element 13 isarranged between the poration tool 6 and the substrate 12 of the objectcarrier 4, which bears the poration tool 6 on its free end movablerelative to the object carrier 4. This has a sharp ring edge 10 on itsend facing away from the piezo element 13, which engages the cell 3 inthe operating position. With its end facing away from the poration tool6, the piezo element 13 is fixed on the substrate 12 of the objectcarrier 4. In the support area 5, the insulator 9 enclosing the porationtool 6 is arranged, which is led over the cone casing surface of theporation tool 6 up to right on its sharp edge 10. By means of the piezoelement 13, the edge 10 of the poration tool 6 can be moved relative tothe object carrier 4 toward or away from a cell 3 positioned on thesupport area 5, approximately in a direction normal to the planestretching across the support area 5. For thiws purpose, the piezoelement 13 is connectable by means of electrical connections 14 with acurrent supply. The insulator 9 consists of an elastic material whichmakes possible a relative motion between the poration tool 6 and thesubstrate 12 of the object carrier 4 during activation of the piezoelement by the connections 14.

[0062] With the piezo element 13, individual mechanical impulses, animpulse succession or a mechanical oscillation can be transmitted to acomponent area of the cell membrane. As a result, an approximatelycircular disk-shaped area of the cell membrane is cut out of themembrane formation of the cell 3. The interior of the cell 3 cansubsequently be manipulated through the opening thereby arising in thecell membrane.

[0063] In the embodiment according to FIG. 7, the poration tool 6 is alaser beam which is launchable through the entry channel 7 into thesupport area 5. The laser beam can be generated with an external laser15 or by means of a laser diode incorporated into the object carrier 4.As is schematically recognizable from FIG. 7, the laser beam is launchedat a site spaced from the mouth of the entry channel 7, by means ofsuitable radiation formation and/or guidance facilities, in thedirection of extension of the entry channel 7 and exits from the supportarea 5 at the mouth of the entry channel 7 approximately in a directionnormal to the surface of the plane stretching across the support area 5.With the laser beam a portion of the cell membrane of the biologicalcell deposited on the support area 5 can be irradiated. The wave lengthof the laser beam is selected so that the cell membrane absorbs thelaser radiation. While the cell membrane is being irradiated with thelaser beam, the membrane is so strongly heated that the cell membraneopens at the irradiated site. After creating the opening, the laserirradiation is turned off, so that the cell interior can then bemanipulated through the opening. The insulator 9 surrounding the mouthof the entry channel 7 in the support area 5 seals the interior of theopened cell adhering to the support area 5 against the culture medium 2.

[0064] In the embodiment according to FIG. 8, a chemical substance isimmobilized around the free end of the poration tool 6 having the sharpedge 10 which protrudes in relation to the surface plane of the supportarea 5 in an approximately annular region. Upon contact with a cell 3deposited on the support area 5, the chemical substance creates anopening in the cell membrane. This embodiment has an especially simpleconstruction.

[0065] In the embodiment in accordance with FIGS. 4 and 5, the entrychannel 7 is filled with a fluid. The entry channel 7 is connected witha pump by which the fluid contained in the entry channel 7 can beimpinged with a controllable overpressure against a cell membrane of acell 3 which is located there and seals off the opening of the entrychannel 7. The edge surrounding the mouth of the entry channel 7 has anannular sharp knife edge 10 which protrudes in relation to the surfaceplane of the support area 5. After depositing the cell 3 on the porationtool 6, a portion of the cell membrane of the cell 3 is impinged bysuction of culture medium 2 from the entry channel 7 for a short time,so strongly with underpressure that the membrane area of the cellmembrane surrounded by the sharp edge 10 of the poration tool 6 isreleased from the membrane formation. In this manner, an opening iscreated in the cell membrane, through which the interior of the cell 3can be manipulated. After creating the opening, the underpressure in theentry channel 7 is shut off.

[0066] After introducing the opening into the cell membrane of the cell3, the pump connected with the entry channel 7 can be reversed for ashort time in the direction of conveyance, in order to inject asubstance situated in the entry channel 7, for example a medicationand/or a fluorescence dye, through the opening of the cell membranedirectly into the cell interior. If no cell 3 is positioned on theporation tool 6, the entry channel 7 can also be used to introduce anappropriate substance into the culture medium 2.

[0067] In the embodiments in accordance with FIGS. 8 and 9, the objectcarrier 4 has several contour depressions 16 respectively running aroundthe poration tool 6 in the support area 5. As is especially wellrecognizable from FIG. 1, the sealing of the cell membrane of the cell 3against the support area 5 of the object carrier 4 is improved.

[0068] The contour depressions 16 are closed annular grooves which arearranged concentrically to the poration tool 6. The annular groovesrespectively have an approximately rectangular cross section. Annulargrooves adjacent to one another are respectively arranged atapproximately identical distances from one another (FIG. 9). Thedistances of adjacent contour depressions 16 to one another and thedepth of these contour depressions 16 are adapted to the type of thecells to be positioned on the support region 5. The edges of the contourdepressions 16 can be rounded in order to facilitate the adherentdepositing of a cell 3.

[0069] The contour depressions 16 can have interruptions in theircourse, as shown with the example of a checkered structuring in FIG. 10and a honeycomb structure in FIG. 11. The surface contours, the surfaceroughness and the surface material, respectively, can be adapted to acertain cell type. In this manner, cell adhesion can be improved orcontrolled.

[0070] In the embodiments according to FIGS. 1 and 8, the surfacecontour 16 is applied on the electrical insulator 9 as a coating usingsemiconductor technology methods. The object carrier 4 is thereby easilyproducible as a semiconductor chip. The surface profiling can, however,also be applied to the insulator 9 with a thick layer technique.

[0071] In the embodiment in accordance with FIG. 8, boundary walls 17are arranged on both sides of the poration tool 6, which together withthe insulator 9 form a guide channel 18 somewhat U-shaped in crosssection. At the same time, the contours 16 and the poration tool 6 arearranged on the floor of the guide channel 18 between the boundary walls17. The boundary walls 17 form an obstacle for cells 3 situated in theguide channel 18, which these cannot overcome, or cannot overcomewithout further effort. The cells 3 can thereby basically moveessentially only in the direction of extension of the guide channel 18,whereby they necessarily come into contact with the poration tool 6.

[0072] The clear distance between the boundary walls 17 arranged on bothsides of the poration tool 6 is adapted to the dimensions of the cells 3and is preferably selected somewhat larger than the diameter of thecells 3. Optionally, several poration tools 6 can be arranged one afteranother in the direction of extension of the guide channel 18. In thismanner, several cells 3 can be opened at the same time and manipulatedintracellularly. The cross section of the guide channel 18 can taper orbroaden in the direction of extension, that is, the guide channel 18 canhave at different places a different width and/or different crosssection dimensions. Proceeding from the deepest to the widest projectingpoint of the guide channel 18, the cross section of the guide channel 18can taper, for example.

[0073] In the embodiment in accordance with FIG. 9, several porationtools 6 are arranged in the form of an array in the support area 5 ofthe object carrier 4. The individual poration tools 6 are respectivelyarranged on the grid points of a Cartesian coordinate system. Theporation tools 6 can, however, also be distributed in another manner inthe support area, for example in rows or columns staggered in relationto each other, or freely distributed.

[0074] Guide structures which enable a selective positioning of cells 3can be arranged on and between the poration tools 6 for optimizing cellgrowth. The guide structures can, for example, include a surfacestructuring, a coating or an appropriate topographical configuration.For various cell types, various distances between poration tools 6adjacent to one another can be provided.

[0075] In the embodiments depicted in the drawings, the entry channel 7is connected respectively with a pump, by means of which culture medium2 is sucked out of the support area 5 of the object carrier 4 and fed atanother spot back to the support area 5. In this manner, the positioningof a cell 3 on the poration tool 6 is facilitated. Optionally, a weakunderpressure can be exerted on the cell 3 after positioning of a cellsucked by means of the entry channel 7 onto the poration tool 6 for acertain time until the cell independently adheres to the support area 5.

[0076] In the embodiments according to FIGS. 1 to 8, the object carrier4 is respectively constructed somewhat plate-shaped. The entry channel 7penetrates the object carrier 4 proceeding from the support area 5 tothe reverse side of the object carrier facing away from this. In theembodiment according to FIG. 1, the object carrier has a wallattenuation 20 which has a smaller wall thickness than the wall arealaterally adjacent thereto. This is obtained by a funnel-shaped recess19 arranged on the reverse side of the object carrier area having thewall attenuation 20. The cross section of the recess 19 narrowsproceeding from the reverse side surface plane of the object carrier 4to the deepest point of the recess 19. A good mechanical stabilityresults therefrom. On the reverse side of the object carrier 4, theentry channel 7 has a connection point 21 for connection with a pump, anunderpressure or over pressure reservoir, a laser or similar facility.It should still be mentioned that in the embodiments according to FIGS.2 to 11, the object carrier can have a reduced wall thickness in thearea of the entry channel 7.

[0077] It will be appreciated by those skilled in the art that changescould be made to the embodiments described above without departing fromthe broad inventive concept thereof. It is understood, therefore, thatthis invention is not limited to the particular embodiments disclosed,but it is intended to cover modifications within the spirit and scope ofthe present invention as defined by the appended claims.

We claim:
 1. A process for intracellular manipulation of at least one biological cell (3) present in a culture medium and adherently deposited on a support area, comprising creating an opening in a membrane of the cell (3) and manipulating the cell interior through this opening, wherein the opening is created in the cell membrane within the support area (5) for the cell (3) and at a distance from an edge of the support.
 2. The process according to claim 1, wherein the opening in the cell membrane is created by sound waves selected from the group consisting of ultrasound and hypersound waves.
 3. The process according to claim 2, wherein the sound waves are focused on an area of the cell membrane to be opened.
 4. The process according to claim 3, wherein several sound waves are superimposed in such a way that their oscillations are superimposed on an oscillation with increased amplitude in the area to be opened.
 5. The process according to claim 1, wherein the cell (3) is fixed on the support area by a force of suction.
 6. The process according to claim 1, further comprising removing or implanting at least one substance and/or cell component from/into the cell interior.
 7. An apparatus for manipulation of a cell interior of at least one biological cell (3) having a cell membrane and being present in a culture medium (2), comprising an object carrier (4) having at least one support area (5) on which the cell (3) is adherently depositable and a poration tool (6) for opening the cell membrane, wherein the poration tool (6) is arranged within the support area (5).
 8. The apparatus according to claim 7, further comprising at least one entry channel (7) located in an area of the poration tool (6) for manipulation of the cell interior
 9. The apparatus according to claim 7, wherein the poration tool (6) within the support area (5) is surrounded by at least one electrical insulator (9).
 10. The apparatus according to claim 8, wherein the poration tool (6) substantially concentrically encloses an opening of the entry channel (7) situated in the support area (5) of the object carrier (4).
 11. The apparatus according to claim 7, wherein the poration tool (6) comprises an electroporation electrode allocated to a reference electrode which can be brought into contact with the culture medium (2), and wherein the electroporation electrode and the reference electrode are connected with a source of electrical voltage for electroporating the cell membrane.
 12. The apparatus according to claim 7, wherein the poration tool (6) is movable relative to the object carrier transversely to the surface of the support area (5) by means of at least one actuator comprising a piezo element (13).
 13. The apparatus according to claim 12, wherein the actuator is connected with an activator facility for generating a mechanical oscillation, including an ultrasound or hypersound oscillation.
 14. The apparatus according to one of claim 7, wherein the poration tool (6) has at least one sharp tip or edge (10) protruding in relation to a surface plane of the support area (5).
 15. The apparatus according to claim 8, wherein the poration tool (b) comprises a laser beam, and the path of the laser beam is guided through the entry channel (7) to the membrane opening situated in the support area.
 16. The apparatus according to claim 15, wherein a laser diode is incorporated into the object carrier (4) for generating the laser beam.
 17. The apparatus according to claim 8, wherein the poration tool (6) comprises a chemical poration substance feedable through the entry channel (7) to an opening therein.
 18. The apparatus according to claim 8, comprising at least one pump for opening the cell membrane by the action of underpressure or overpressure, the pump being connected with the entry channel (7) and an underpressure or overpressure reservoir through a shut-off element.
 19. The apparatus according to claim 7, wherein the poration tool (6) is arranged on a projection (11) protruding in relation to a surface plane of the support area (5).
 20. The apparatus according to claim 19, wherein a cross section of the projection (11) tapers proceeding from the surface plane of the support area (5) to a furthest protruding point.
 21. The apparatus according to claim 7, wherein the object carrier (4) has a profile in the support area (5) which has at least one contour depression running around the poration tool and/or a contour projection (16) running around the poration tool.
 22. The apparatus according to claim 21, wherein the contour depression and/or contour projection (16) is interrupted by at least one gap in a direction of extension of the object carrier.
 23. The apparatus according to claim 21, wherein the contour depression and/or the contour projection (16) is constructed ring-shaped with several annular contour depressions and/or contour projections (16) arranged substantially concentrically to the poration tool (6).
 24. The apparatus according to claim 9, wherein the electrical insulator (9) comprises an insulation layer arranged on a contoured surface.
 25. The apparatus according to claim 21, wherein contour projection(s) is (are) applied to a surface of an electrical insulator.
 26. The apparatus according to claim 7, further comprising in the support area (5) of the object carrier (4), a coating having at least one cellular adhesion protein and/or a hydrophilic coating and/or a hydrophobic coating arranged on a surface of the support area directly adjacent to the poration tool.
 27. The apparatus according to claim 8, wherein the object carrier (4) is constructed as an approximately plate-shaped carrier element which has the support area (5) on a flat side, the entry channel (7) penetrates the object carrier (4) proceeding from the support area (5) to the reverse side of the object carrier (4), and the object carrier has a wall attenuation (20) in an area of the entry channel (7), which has a smaller dimension in a direction of extension of the entry channel (7) than an adjacent wall area adjacent to the wall attenuation.
 28. The apparatus according to claim 27, wherein the wall attenuation (20) is arranged on a funnel-shaped recess (19) situated on a reverse side of the object carrier (4) facing away from the support area (4).
 29. The apparatus according to claim 7, wherein boundary walls (17) are arranged on both sides of the poration tool (6) as a mechanical guide for the cells (3) and the walls (17) delimit a groove-shaped guide channel (18).
 30. The apparatus according to claim 7, further comprising at least one supplemental electrode arranged in or adjacent to the support area (5) for generating an electric field leading the cell (3) to the poration tool (6).
 31. The apparatus according to claim 8, wherein the support area (5) has several poration tools (6) having at least one entry channel (7) respectively arranged as arrays. 